Despite significant progress in diagnostics and disease management during the past decades, human immunodeficiency virus (HIV) infections are still responsible for nearly 1 million deaths every year, mostly in resource-limited settings. Thus, novel, accurate and cost-effective tools for viral load monitoring become crucial to allow specific diagnostics and the effective monitoring of the associated antiviral therapies. Herein, we report an effective combination of a (1) padlock probe (PLP)-mediated rolling circle amplification (RCA) bioassay and an (2) agarose bead-based microfluidic device for the affinity chromatography-based capture and detection of RCA products (RCPs) pre-labelled simultaneously with biotin and an organic fluorophore. This method allowed the efficient capture of similar to 1 mu m-sized RCPs followed by their quantification either as discrete signals or an average fluorescence signal, thus being compatible with both high-resolution imaging for maximum sensitivity as well as simpler optical detection setups. A limit of detection < 30 fM was obtained for HIV-1 synthetic target with just a single round of RCA, comparable to recently reported procedures requiring technically complex amplification strategies such as hyperbranching and/or enzymatic digestion/amplification. Furthermore, targeting a set of five conserved regions in the HIV-1 gag gene, the method could specifically detect HIV-1 in 293T cell culture supernatants, as well as a set of 11 HIV-1 NIH reference samples with four different subtypes. The reported method provides simplicity of operation, unique versatility of signal transduction (i.e. average or discrete signals), and potential coupling with previously reported miniaturized photodetectors. These combined features hold promise for bringing RCA-based molecular diagnostics closer to the point-of-care.